Avidity of anti-glomerular basement membrane autoantibodies was associated with disease severity

Anti-glomerular basement membrane (GBM) antibody-mediated diseases are characterized by the binding of autoantibodies to GBM, leading to rapidly progressive glomerulonephritis that often results in irreversible loss of renal function. The nephrotoxic potential of anti-GBM antibodies has been demonstrated in animal experiments. We questioned whether high avidity leads to persistent deposition of anti-GBM antibodies, thereby perpetuating inflammation and renal damage. To address the hypothesis, sera from 32 patients and serial samples from 11 patients with anti-GBM disease were collected. Purified bovine alpha chain non-collagen 1 domains of type IV collagen [alpha(IV)NC1] were employed to exam avidity of anti-GBM antibodies using antigen-inhibition enzyme-linked immunosorbent assay. The amount of alpha(IV)NC1 needed for 50% inhibition of antibody binding was compared among patients with different clinical and pathological parameters. After the sera were diluted to give equivalent concentration of anti-GBM antibodies, the amount of alpha(IV)NC1 used for 50% inhibition was prominently different among patients, from 0.02 microg to 20 microg, with an average at 0.666 microg. A significant correlation was observed between the amount of alpha(IV)NC1 used and the percentage of glomeruli which had crescents (P=0.001). Higher avidity of anti-GBM antibodies predicted higher percentage of glomerular crescents (R2=0.58, P<0.001). No obvious change of avidity was observed in the serial samples. The results suggested that affinity maturation might have been completed by the time that patients presented with anti-GBM disease. The avidity of anti-GBM antibodies was associated with the degree of renal damage and might play a key role in the pathogenesis of anti-GBM disease.     

Anti-GBM glomerulonephritis: a T cell-mediated autoimmune disease?

Anti-glomerular basement membrane (GBM) glomerulonephritis, which was among the earliest recognized human autoimmune diseases, is characterized by the presence of anti-GBM antibody. It has been a prototypical example of autoantibody-mediated autoimmune disease. However, decades of research on this disease, based either on clinical observations or experimental models, have revealed that T cell-mediated cellular immunity may potentially be a more important mediator of glomerulonephritis. We have made several breakthroughs in understanding the T cell-mediated mechanism causing this disease in a rat model based on Goodpasture's antigen, non-collagen domain 1 of alpha3 chain of type IV collagen (Col4alpha3NC1). We demonstrated that anti-GBM glomerulonephritis was induced by either passive transfer of Col4 alpha3NC1-specific T cells or active immunization with the nephritogenic T cell epitope of Col4alpha3NC1. Immunization with the T cell epitope also triggered production of anti-GBM antibodies to diversified GBM antigens. Thus, a single nephritogenic T cell epitope alone is sufficient to induce the clinical spectrum of anti-GBM glomerulonephritis, including proteinuria, glomerular injury, and anti-GBM antibody. A possible T cell-mediated mechanism for causing human anti-GBM disease is proposed.     

Serial functional affinity of autoantibodies in anti-glomerular basement membrane disease.

Anti-glomerular basement membrane (GBM) disease is caused by an autoantibody directed against an epitope on the alpha 3 chain of type IV collagen. Animal models demonstrate that the higher the affinity of such antibodies, the greater the degree of glomerular injury. Affinity maturation (the process whereby somatic mutation followed by antigen selection leads to an increase in affinity of antibody) might therefore be of pathogenic significance if it occurs in human anti-GBM disease. We have examined serial samples from nine patients with anti-GBM disease and looked for evidence of changing functional affinity by measuring the inhibition of binding produced by the mild chaotrope diethylamine (DEA) in an anti-GBM antibody ELISA. Seven patients showed no change in the inhibition produced by DEA with time. Two patients showed an apparent decrease with time in the inhibition produced by DEA; this apparent increase in functional affinity proved, on further investigation, to represent simply the loss of anti-GBM antibodies. These results may imply that affinity maturation has been completed by the time that patients present with anti-GBM disease. If there had been evidence for a further increase in functional affinity after this point then this might have added extra urgency to the need for removal of these autoantibodies as part of treatment.     

Natural anti-GBM antibodies from normal human sera recognize alpha3(IV)NC1 restrictively and recognize the same epitopes as anti-GBM antibodies from patients with anti-GBM disease

Anti-GBM disease is a rare autoimmune condition characterized by autoantibodies targeting the alpha3 chain non-collagen 1 domain of type IV collagen (alpha3(IV)NC1). Recently, we isolated IgG reacting with alpha3(IV)NC1 from normal healthy human sera. The current study examined the antigen and epitope specificity of these natural autoantibodies (NAA) using recombinant human alpha1, 3, 5(IV)NC1 and three constructs expressing, previously defined epitope regions designated E(A), E(B) and S2, in the alpha1(IV)NC1 background. The NAA preparations reacted with recombinant human alpha3(IV)NC1 to the same extent as with purified bovine alpha(IV)NC1, but not with recombinant human alpha1 and alpha5(IV)NC1. NAA preparations recognized the three chimeric proteins (E(A), E(B) and S2) yielding similar absorbance values. We conclude that anti-GBM NAA recognize the same major epitopes as anti-GBM antibodies from patients with Goodpasture's disease.     

Characterization of monoclonal antibodies to the globular domain of collagen IV.

Monoclonal antibodies were produced against NC1, the globular noncollagenous domain of collagen IV, isolated from bovine glomerular basement membrane. Cells from eight positive wells were cloned and the resulting monoclonal antibodies were studied in detail by immunofluorescence on human kidney sections, by Western blot and by ELISA against denatured subunits from NC1 hexamers and against native NC1 hexamers from different tissues. The monoclonal antibodies could be divided into two groups. Firstly, those monoclonal antibodies that, in ELISA and Western blot, reacted with peptides related to the alpha 1 chain of collagen IV and stained all basement membranes in the kidney. Secondly, a monoclonal antibody that, in ELISA and Western blot, reacted with peptides related to the Goodpasture antigen, the alpha 3 chain of collagen IV. When this antibody was applied to human kidney sections it stained the glomerular basement membrane very intensively. Bowman's capsule and some tubular basement membrane were also stained, although to a lesser extent. This staining pattern is the same as that observed with sera from patients with Goodpasture's syndrome. An attempt was made to separate different subtypes of the NC1 hexamer. A monoclonal antibody from the first group was used to make an affinity chromatography column. Glomerular basement membrane digested with collagenase was separated on this column and the collected fractions were analyzed by ELISA and SDS-PAGE. The result from this study support the idea that glomerular basement membrane is composed of at least two different subtypes of type IV collagen.     

The non-collagenous domains of the alpha 3 and 4 chains of type IV collagen and their relationship to the Goodpasture antigen

The Goodpasture antigen is the target recognized by anti-glomerular basement membrane (GBM) antibodies in anti-GBM disease or Goodpasture's syndrome. This structure is present in all normal GBM, but when serum containing anti-GBM antibodies is used to examine renal tissue from most males with classical Alport's syndrome, the Goodpasture antigen appears to be missing. The nature of the Goodpasture antigen is uncertain although it has been putatively and controversially localized to the non-collagenous domain of a novel type IV collagen chain (alpha 3) by one group, and a short peptide sequence has been published (M2). We have performed several experiments to determine whether M2 represents the Goodpasture antigen and we have also studied the corresponding sequence of the alpha 4 chain of type IV collagen (M3). Firstly, we demonstrated by polymerase chain reaction (PCR) amplification using specific priming oligonucleotides that mRNAs corresponding to M2 and M3 were found within the kidney and that the published sequences were correct. When heterologous antibodies were raised against M2 and M3 these bound specifically to GBM in an ELISA based on collagenase-digested basement membrane and this binding could be inhibited by incubation with collagenase-digested GBM but not with ovalbumin. On further examination of the target molecules using Western blots, the anti-M2 antibody bound to a single high molecular weight band of collagen-digested GBM in contrast to the anti-M3 antibody that bound to the same bands as Goodpasture serum. We then established ELISAs for anti-M2 and anti-M3 activity using the peptides M2 and M3. While rabbit anti-M2 and M3 antibodies bound specifically to their respective peptides in these ELISAs, there was no binding of three high titre Goodpasture's syndrome sera or two sera from Alport's syndrome patients with inhibitable anti-GBM antibody post-renal transplant. We have shown that the sequences of M2 and M3 correspond to proteins present within the collagenase-resistant part of the GBM, suggesting that these do represent parts of novel type IV collagen chains. However, sera containing anti-GBM antibodies did not bind to either peptide in solid-phase ELISAs, and these antibodies may recognize a different peptide sequence, features of the tertiary structure of these peptides or interactions between collagen chains.     

Restricted specificity of the autoantibody response in Goodpasture''s syndrome demonstrated by two-dimensional western blotting.

The autoantigen in Goodpasture's syndrome is known to be contained within the non-collagenous (NC1) domain of type IV collagen. We have examined the specificity of autoantibodies to glomerular basement membrane (GBM) using the technique of 2-D electrophoresis followed by Western blotting. Protein stains of 2-D gels of collagenase-digested human GBM revealed extensive charge and size heterogeneity. Major components were of mol. wt 24-30 kD and 43-56 kD, corresponding to monomeric and dimeric subunits of NCl. Western blotting of 2-D gels with IgG from patients with anti-GBM disease demonstrated that the most antigenic components migrated as cationic 28-kD monomers (pI 10) and similarly charged dimers, although other components were recognized less strongly. The mobility of the strongly antigenic polypeptides was different to that of the known alpha 1 and alpha 2 chains of type IV collagen. Autoantibodies from all 20 patients studied showed the same pattern of reactivity, regardless of their clinical features (in particular, the presence or absence of pulmonary haemorrhage) or HLA type. A monoclonal antibody (P1) to human GBM bound in a similar pattern, particularly recognizing the cationic components. 2-D gels of affinity-purified GBM from a P1 column showed enrichment of the 28-kD monomers, which were recognized by human autoantibodies on Western blotting. These results demonstrate that the autoimmune response in Goodpasture's syndrome is of restricted specificity, and support the suggestion that the major autoantigenic determinant is present on the novel alpha 3 chain of type IV collagen.     

Properties of HLA class II molecules divergently associated with Goodpasture's disease

Goodpasture's disease provides an opportunity to analyse molecular mechanisms that may underlie MHC class II associations with autoimmune disease because it is caused by autoimmunity to a defined antigen [the 230 amino acid NC1 domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1)] and has strong HLA class II associations. We compared the alpha3(IV)NC1 peptide binding of class II molecules with strong positive (DR15) and dominant negative (DR7/1) associations using an inhibition binding assay and short synthetic peptides spanning the sequence of alpha3(IV)NC1. DR15 in general bound the peptides with low affinity (three of 23 < 100 nM) compared to DR1 and DR7 (12 and 10 < 100 nM respectively), and no peptide bound DR15 with much higher affinity (>10-fold) than both DR1 and DR7. Thus DR15 molecules are unlikely to increase susceptibility to Goodpasture's disease by presenting a particular alpha3(IV)NC1-derived peptide uniquely well and DR1/7 are unlikely to protect by their inability to present particular peptides. However DR1/7 could protect by capturing alpha3(IV)NC1 peptides and preventing their display bound to DR15; the binding data suggest that all the major (biochemically detectable) alpha3(IV)NC1 peptides presented bound to DR15 by DR15 homozygous antigen-presenting cells (APC) would bind preferentially to DR1/7 in DR15, 1/7 heterozygote APC.     

Genetic susceptibility to experimental autoimmune glomerulonephritis in the Wistar Kyoto rat

In experimental autoimmune glomerulonephritis (EAG), a model of Goodpasture's disease, Wistar Kyoto (WKY) rats immunized with collagenase-solubilized glomerular basement membrane (GBM) or the recombinant NC1 domain of the α3 chain of type IV collagen [α3(IV)NC1] develop anti-GBM antibodies and focal necrotizing glomerulonephritis with crescent formation. However, Lewis (LEW) rats, which share the same major histocompatibility complex (MHC) haplotype, are resistant to EAG development. A genome-wide linkage analysis of backcrossed animals with EAG revealed a major quantitative trait locus (QTL) on rat chromosome 13 (LOD = 3.9) linked to the percentage of glomerular crescents. To investigate the role of this QTL in EAG induction, reciprocal congenic rats were generated (LEW.WCrgn1 congenic and WKY.LCrgn1 congenic), immunized with recombinant rat α3(IV)NC1, and assessed for EAG development. WKY.LCrgn1 rats showed a marked reduction in albuminuria, severity of crescentic nephritis, and number of glomerular macrophages compared with WKY controls. No reduction in antibody levels was observed. However, LEW.WCrgn1 rats were resistant to EAG development, as were LEW controls. Macrophage activation in vitro was assessed in parental and congenic rat bone marrow-derived macrophages (BMDMs). WKY.LCrgn1 BMDMs showed a significant reduction in Fc receptor-mediated oxidative burst, phagocytosis of opsonised polystyrene beads, and LPS-induced levels of MCP-1 secretion and iNOS mRNA expression compared with WKY rats. These results confirm the importance of Crgn1 on chromosome 13 in EAG susceptibility, mediated partly through differences in Fc receptor-mediated macrophage activation.     

A single autoantigen in Goodpasture's syndrome identified by a monoclonal antibody to human glomerular basement membrane

A mouse monoclonal antibody (P1) to the autoantigenic component of human glomerular basement membrane (GBM) was used to study the immunochemistry and tissue distribution of the Goodpasture antigen and the specificity of the human autoimmune response in Goodpasture's syndrome (anti-GBM disease). In solid phase assays, monoclonal antibody P1 bound to collagenase-solubilized human GBM (the ligand used in assays for human autoantibody), but not to other biochemically defined components of basement membrane. On Western blotting, P1 bound to the same 6 bands in solubilized GBM (between 26 and 58 kilodaltons with major bands at 26 and 54 kilodaltons) that were recognized by sera from all 42 patients studied with anti-GBM disease. Preincubation with sera from 8/8 patients blocked the subsequent binding of P1 from 83 to 89% on densitometer scanning of the Western blot; and preincubation with P1 blocked the binding of sera from 6/6 patients from 58 to 89%. Indirect immunofluorescence and immunoperoxidase studies revealed that the pattern of binding of P1 was identical to that of antibody eluted from the kidneys of a patient with Goodpasture's syndrome; there was linear binding to GBM, Bowman's capsule, and distal tubular basement membrane. In addition, P1 bound to basement membranes in lung and choroid plexus, and to membranes of the lens capsule, choroid, and retina of the eye and cochlea, but not to other organs studied. It is concluded that there is a single major autoantigenic component of human GBM (the Goodpasture antigen), which is present on fragments of different molecular weight in the collagenase digest. This antigen is distributed throughout well-defined basement membranes known to be involved in both Goodpasture's and Alport's syndromes. Human anti-GBM antibodies bind to the same (or closely related) determinants which are recognized by P1, demonstrating that the autoimmune response in Goodpasture's syndrome is of highly restricted specificity.     

Epitope mapping of anti-glomerular basement membrane (GBM) antibodies with synthetic peptides

Autoantibodies to the non-collagenous (NC1) domain of the α3(IV)-chain of type IV collagen are found in sera from patients with anti-GBM nephritis. These antibodies have been shown to be pathogenic. In this study the antibody specificity has been investigated in patients with Goodpasture’s syndrome and from a patient with atypical anti-GBM antibodies, recognizing the α1(IV)-chain only. Overlapping synthetic peptides, covering the complete NC1 domains of the α1(IV)- and α3(IV)-chains were used in sandwich ELISA and competitive ELISA. None of the Goodpasture sera showed reactivity to the synthetic peptides. However, antibodies from the patient with atypical anti-GBM antibodies recognized a 20 amino acid peptide from the α1(IV)-chain. The reactive peptide was further narrowed down with glycine substitution of the different amino acids. We have localized the epitope to the four last C-terminal amino acids of the α1(IV)-chain, with the sequence 1754-MRRT. The two arginine residues were found to be essential for antibody binding. Threonine is important, while methionine is of less importance. These four amino acids are also determined to be the smallest peptide that could inhibit the binding of the autoantibodies to the native α1(IV)-chain. This study shows that overlapping peptides can be used to map linear epitopes. However, for conformational epitopes such as the Goodpasture epitope, other methods must be used. It would be prognostically important to know the fine specificity of anti-GBM antibodies, since the patient with anti-α1(IV) antibodies had a mild disease, while the Goodpasture patients with anti-α3(IV) antibodies had a rapidly progressive disease.     

A study by immunofluorescence microscopy of the NC1 domain of collagen type IV in glomerular basement membranes of two patients with hereditary nephritis

Summary The NC1 domain of the collagen type IV molecule, the major component of glomerular basement membranes (GBM), consists of dimers and 24 kilodalton (K), 26 K and 28 K monomers in man, and contains the Goodpasture antigen. Serum obtained from patients with Goodpasture's syndrome has been reported not to stain GBM of most male and some female patients with hereditary nephritis (HN) by immunofluorescence (IF) microscopy. In the present study, GBM seen on the renal biopsies of 2 patients (one male and one female) with HN were examined by IF to ascertain whether NC1 monomers were detectable. Three reagents were used: a plasmapheresis fluid (PPF) obtained from a patient who was treated for anti-GBM nephritis (human anti-GBM PPF); a commercial rabbit antibody against human NC1; and a rabbit antibody raised by us against dog NC1, which cross-reacted with human NC1. All 3 reagents detected NC1 determinants in GBM of normal human kidney by IF and reacted with human NC1 by a plate-binding radioimmunoassay (RIA). The human anti-GBM PPF bound to 28 K and 26 K monomer components of NC1 by Western blotting, the rabbit anti-human NC1 antibody bound to 26 K and 24 K monomers, while the rabbit anti-dog NC1 antibody bound only to the 26 K monomer. By IF, the human anti-GBM PPF did not stain GBM of the male patient with HN, but produced segmental staining of GBM (i.e., some GBM stained, while others did not) of the female patient. In contrast, the rabbit anti-NC1 antibodies produced global staining by IF of GBM of both patients. The absence of staining (i.e., global or segmental) seen with the human anti-GBM PPF implied that the 26 K and 28 K monomers of NC1 were either absent from GBM, or were present but altered structurally, leading to a diminution in their immunological reactivity. However, the positive staining observed with the rabbit anti-NC1 antibodies implied that the 26 K monomer was actually present in GBM. Hence, we postulate that the 26 K monomer of NC1 in GBM was structurally altered, and that the 28 K monomer was either absent, or present but altered. These findings suggest that there is an abnormality of more than one monomer of NC1 in GBM of patients with HN.     

Antimitochondrial autoantibodies in anti-glomerular basement membrane disease.

Anti-glomerular basement membrane (GBM) disease is characterized by the production of an autoantibody with very restricted specificity, with no evidence of polyclonal B cell activation. It was therefore surprising to find that in a solid-phase ELISA a proportion of anti-GBM sera showed significant binding to pyruvate dehydrogenase (PDH), a reactivity usually associated with the antimitochondrial autoantibodies (AMA) found in primary biliary cirrhosis (PBC). The specificity of this reactivity was confirmed by inhibition and competition experiments. The AMA found in anti-GBM sera were of much lower affinity than those found in PBC sera, and recognized a more restricted set of species (mainly the 55-kD and occasionally the 74-kD component of PDH). However, it was possible to block the binding in a Western blot of an anti-GBM serum to both the 55-kD and 74-kD species with F(ab')2 fragments prepared from a PBC serum. Although AMA have been found in diseases other than PBC, such diseases have usually been characterized by polyclonal B cell activation. The stimulus to the production of AMA in anti-GBM disease, and their significance in pathogenesis (if any), are unknown.     

Leukocyte involvement in the pathogenesis of pulmonary injury in experimental Goodpasture's syndrome

To investigate the role of leukocytes in the pathogenesis of pulmonary injury after antibody deposition on the alveolar basement membrane, we have induced experimental Goodpasture's syndrome in the rat using passive accelerated anti-glomerular basement membrane (GBM) disease. Animals were immunized with normal rabbit IgG and given either rabbit anti-rat GBM serum or normal rabbit serum (control) 5 days later. In this model, leukocytic infiltration, severe hemorrhage, and granulomatous-like lesions developed in the lung, while a rapidly progressive glomerulonephritis was evident in the kidney. Within 30 minutes of injection of anti-GBM serum, strong linear deposition of antibody on both glomerular and alveolar basement membranes was evident. In the lung, a transient influx of polymorphonuclear cells during the first 12 hours was closely followed by macrophage infiltration, with T cell infiltration not evident until day 3. Pulmonary hemorrhage correlated with ED1+ macrophage infiltration (p less than 0.001), but not with OX-19+ T cell accumulation. Many activated mononuclear cells were found in the lung infiltrate from day 14 onward and were associated with areas of tissue damage. This study demonstrates the feasibility of using passive accelerated anti-GBM disease as a model of Goodpasture's syndrome, and suggests that inflammatory macrophages are the major cellular participants in progressive pulmonary injury after antibody deposition.     

Advances in the pathogenesis of Goodpasture's disease: from epitopes to autoantibodies to effector T cells

Goodpasture's disease, an "organ-specific" autoimmune disease is manifest by rapidly progressive glomerulonephritis and pulmonary hemorrhage. Studies into the pathogenesis of this disease have shed light on the autoantigen (the non-collagenous domain of the alpha3 chain of type IV collagen, alpha3(IV)NC1) and its epitopes, as well as the involvement of autoantibodies and cellular effectors in disease. The discovery of alpha3(IV)NC1 lead to studies that defined the structure and biology of type IV collagen and are defining B and T cell epitopes. Goodpasture autoantibody epitopes are "cryptic" in that they are structurally sequestered by adjacent non-collagenous domains of alpha4 and alpha5 type IV collagen. T cell epitope studies in rats demonstrated that a 13-mer could induce experimental autoimmune glomerulonephritis. T cells from patients with Goodpasture's recognize two epitopes, in regions which are highly susceptible in antigen processing by endosomal proteases. Goodpasture's disease is strongly associated with HLA DRB1 genes, whereby DRB1*1501 confers susceptibility and the DRB1*0701 and DRB1*0101 are dominantly protective. Experimental data implicate both autoantibodies and cell mediated immunity as disease effectors. Observations in humans suggest that regulatory T cells are associated with the development of self-immunoregulation in the convalescent phase of disease.     

Glomerulonephritis with focal and segmental distribution of glomerular basement membrane antigen(s)

In this report, the authors describe a case of a 2-year, 11-month-old girl with glomerulonephritis and no family history of renal diseases and deafness. Immunofluorescent studies in the renal biopsy specimens with the use of anti-sera against human glomerular basement membrane (GBM) and P3 antigen (prepared from bovine GBM and inducible of Steblay's type nephritis in rats) demonstrated focal and segmental distribution of the GBM antigen(s). Electron microscopic examination revealed splitting and thinning of the GBM. Indirect immunofluorescence showed that there was no binding of Goodpasture's anti-GBM antibodies to the glomeruli. These findings are similar to those in patients with hereditary nephritis. The immunofluorescent examination of the fixation of the various anti-sera, including anti-types IV and V collagens, laminin, fibronectin, and actomyosin sera on the GBM, revealed normal reactivity. The abnormalities observed in this case may be a part of the spectrum of primary GBM defects.     

The evolution of crescentic nephritis and alveolar haemorrhage following induction of autoimmunity to glomerular basement membrane in an experimental model of Goodpasture's disease

Goodpasture's, or anti-glomerular basement membrane (GBM), disease presents with rapidly progressive glomerulonephritis and lung haemorrhage, and is caused by autoimmunity to the NC1 domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). This study examines the development of crescentic nephritis and alveolar haemorrhage in a model of Goodpasture's disease, experimental autoimmune glomerulonephritis (EAG), induced in WKY rats by immunization with rat GBM in adjuvant. An increase in circulating anti-GBM antibodies and albuminuria was observed by week 2, which increased further by weeks 3 and 4, while a decrease in creatinine clearance was observed by week 2, which decreased further by weeks 3 and 4. The kidneys of animals with EAG showed linear deposits of IgG on the GBM and a transient glomerular infiltration by CD4+ T cells at week 2. By week 3 there were large deposits of fibrin in Bowman's space, and glomerular infiltration by CD8+ T cells and macrophages, accompanied by focal necrotizing glomerulonephritis with crescent formation. Ultrastructural studies showed glomerular endothelial cell swelling and epithelial cell foot process effacement at week 2. As the lesion progressed, capillary loops became occluded and the mesangium became expanded by mononuclear cells. By week 3 there was detachment of the endothelium from the GBM, and accumulation of fibrin beneath the disrupted endothelial cells and in Bowman's space. Occasional breaks were observed in the continuity of the basement membrane, and cytoplasmic projections from infiltrating mononuclear cells could be seen crossing the capillary wall between the lumen and the crescent. The lungs of animals with EAG showed patchy binding of IgG to the alveolar basement membrane (ABM) at week 2, and infiltration of the interstitium by CD8+ T cells and macrophages by weeks 3 and 4, accompanied by both interstitial and alveolar haemorrhage. Ultrastructural studies showed focal mononuclear cell infiltrates in alveolar walls at week 2. Occasional breaks were observed in the basement membrane and adjacent endothelium by weeks 3 and 4, together with accumulation of surfactant and erythrocytes within the alveolar spaces. This study defines for the first time the relationship between the immunological and pathological events during the evolution of EAG, and provides the basis for further work on the pathogenesis of Goodpasture's disease.     

Comparison of non-collagenous type IV collagen subunits in human glomerular basement membrane, alveolar basement membrane, and placenta

This study examines the similarities and differences in the noncollagenous domain (NC1) of type IV collagen from human glomerular basement membrane (hGBM), alveolar basement membrane (hABM), and placenta (hPBM). Following collagenase digestion, NC1 domain was isolated on Bio-Gel A-0.5m or by cation exchange chromatography on S-Sepharose. NC1 from each source was characterized by SDS PAGE, and two dimension NEPHGE/SDS PAGE. Immunoblotting and ELISA inhibition was performed using antibody probes specific for M28 , M28+, M26 and M24 monomer subunits of human NC1. It was observed that all NC1 subunits were present in hGBM and hABM derived material, however M28 and M28+ monomers were absent in hPBM NC1. These findings indicate that while alpha 1(IV) and alpha 2(IV) collagen chains are present in hGBM, hABM and hPBM, alpha 3(IV) and alpha 4(IV) collagen chains are only found in hGBM and hABM but are absent in hPBM. It can now be appreciated that heterogeneity of alpha (IV) chain composition exists in basement membranes from various organs.     

Effect of glomerular basement membrane on the initiation of chemiluminescence and lysosomal enzyme release in human polymorphonuclear leucocytes: an in vitro model of glomerular disease

The effect of glomerular basement membrane (GBM) on the stimulation of human polymorphonuclear leucocytes (PMNL) was investigated using the technique of chemiluminescence (CL). GBM, in the absence of serum, did not initiate CL of PMNL and neither did GBM preincubated with fresh human or rabbit serum. In contrast, GBM preincubated with rabbit anti-human GBM serum (anti-GBM), and then thoroughly washed, initiated an immediate and marked CL response. Heat inactivation of the anti-GBM only partially reduced the CL, suggesting that both Fc and C3b receptors on the PMNL were involved. The addition of fresh, but not heat-inactivated, human serum to PMNL incubated with untreated GBM resulted in a CL response after a short lag. Subsequent analysis of the serum by electrophoresis and immunofixation indicated that the GBM had activated complement. GBM preincubated with anti-GBM was found to elicit the selective release of lysosomal enzymes from PMNL, one of these enzymes degrading the collagen moiety of the GBM. Preincubation of GBM with the serum from a patient with Goodpasture's syndrome also initiated a CL response and a selective release of lysosomal enzymes from PMNL. These results provide additional evidence for imputing a role for PMNL in the pathogenesis of glomerulonephritis. Furthermore, the technique offers a useful in vitro model for the study of immunologically mediated glomerular disease.     

Recombinant α-chains of type IV collagen demonstrate that the amino terminal of the Goodpasture autoantigen is crucial for antibody recognition

Goodpasture's disease, an autoimmune disorder causing severe glomerulonephritis and pulmonary haemorrhage, is characterized by antibodies to the glomerular basement membrane (GBM). The principal target antigen has been identified as the carboxyl terminal non-collagenous (NC1) domain of the α3-chain of type IV collagen. Anti-GBM antibodies appear to recognize one major epitope that is common to all patients, and is largely conformational. We have analysed antibody binding to recombinant α(IV)NC1 domains using a construct and expression system shown to produce correctly folded antigen that is strongly recognized by autoantibodies. In this system, as with the native antigen, α3(IV)NC1 was bound strongly by antibodies from all patients, whereas the closely related α1(IV) and α5(IV)NC1 domains, similarly expressed, showed no such binding. A series of chimeric NC1 domains, between human α3(IV) and α1(IV), and between human and rat α3(IV), were expressed as recombinant molecules, and were recognized by autoantibodies to varying degrees. Strong binding required the presence of human α3(IV) sequence in the amino terminal region of both sets of chimeric molecules. This work strongly suggests that the amino terminal of α3(IV)NC1 is critical for antibody recognition, whereas the carboxyl terminal end of α3(IV)NC1 has a less important role.